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In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.

We have stabilized and equalized field-emission (FE) currents from carbon nanotube (CNT) emitters by a cascade active-current-control (ACC) using two transistors. Theoretical and experimental analyses on the cascade ACC operation, including single ACC with a single transistor, were done for both diode- and triode-configured CNT emitters. A fully vacuum-sealed CNT FE X-ray tube (FEXT) was used as a triode-configured test device. FE currents, in both configurations, by the cascade ACC were found to be stabilized to a standard deviation of 0.05% even in high-voltage and high-power circumstances while the single ACC could hardly regulate fluctuations in FE currents from CNT emitters. Furthermore, an array of CNT FEXTs successfully demonstrated a completely equalized emission current when the cascade ACC was employed. This result has important implications for FEXT current uniformity and other FEXTs applications, because the cascade ACC can equalize FE currents even through large variation of CNT emitters, thereby greatly enhancing the usability of FE devices.

Aims The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs. Methods and results In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1β-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs. Conclusion Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.

Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogene-free hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes. These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders.

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1–10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p &lt, 0.05) and upregulated intracellular glutathione levels (p &lt, 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p &lt, 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0–2), while significantly (p &lt, 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p &lt, 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

Simple Summary V-set and immunoglobulin domain-containing 1 (VSIG1) is a newly discovered member of the junctional adhesion molecule (JAM) family encoded by a gene located on the X chromosome in humans and mice. Expression of VSIG1 in normal mammalian tissues was originally reported to be highly tissue-specific and was detected in organs such as the testis. However, as little is known about its physiological function, we aimed to investigate the function of VSIG1 in mammalian spermatogenesis and fertilization. Abstract To elucidate the functional role of V-set and immunoglobulin domain-containing 1 (VSIG1) in spermatogenesis and fertilization, we knocked out (KO) VSIG1 in a mouse embryo using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) -mediated genome editing. Reverse transcription PCR was performed using cDNA synthesized from VSIG1 KO testis RNA. Although Western blot analysis using a specific antibody to VSIG1 confirmed VSIG1 protein defects in the KO mice, hematoxylin-eosin staining analysis was similar in the KO and wild-type mice. Additionally, computer-assisted sperm analysis and in vitro fertilization experiments were conducted to confirm the activity and fertilization ability of sperm derived from the KO mouse. Mice lacking VSIG1 were viable and had no serious developmental defects. As they got older, the KO mice showed slightly higher weight loss, male mice lacking VSIG1 had functional testes, including normal sperm number and motility, and both male and female mice lacking VSIG1 were fertile. Our results from VSIG1 KO mice suggest that VSIG1 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. VSIG1 in sperm is dispensable for spermatogenesis and male fertility in mice. As several genes are known to possess slightly different functions depending on the species, the importance and molecular mechanism of VSIG1 in tissues of other species needs further investigation.

Background The liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male’s liver are good in alcohol clearance and lipid metabolism, while female’s liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently. Methods Mouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model. Results In this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex. Conclusion In conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

Fluoroscopy is a radiological technique that provides real-time x-ray viewing in interventional and angiographic procedures. In fluoroscopic procedures, there are several issues have to be solved, such as a risk of radiation exposure to the patients and operators and low image qualities by motion blur. To lower the radiation dose and motion blur, most of fluoroscopic systems provide a pulse-mode operation. However, conventional systems adopt filament-based thermionic analog x-ray tubes that generate relatively longer x-ray pulses than a few milliseconds due to intrinsic difficulty in modulating electron emissions, thus still have many problems of motion blur for fast objects, unnecessary x-ray radiation, and mismatched frame rate to the moving objects. In this work, we tried to solve these problems by suggesting an adaptively variable frame-rate fluoroscopy with an ultra-fast digital x-ray tube (DXT) based on carbon nanotube (CNT) field electron emitters. We first fabricated a vacuum-sealed CNT DXT and its monoblock with a power generator for the fluoroscopic system. Ultra-short and high-frequency x-ray pulses of up to 500 ns at 1 MHz was achieved by the direct control of electron emission through an active current-control unit. X-ray pulse frames from the CNT DXT with a tube voltage of 120 kV and current of 20 mA were adaptively modulated in the range of 1-30 Hz according to the motion of objects, greatly improving temporal resolution with a reduced radiation dose. The adaptively variable frame-rate fluoroscopy could pave the way for both reducing x-ray doses and improving temporal and spatial resolution.

Simple Summary To increase the early implantation rate, oocytes and zygotes have been subjected to various artificial stimulations before and/or after in vitro fertilization, nuclear transfer, or sperm (spermatid) injection, etc. However, the stimulation process may induce parthenogenetic development. It is difficult to identify whether the embryo or fetus is normally fertilized or parthenogenetically activated in early pregnancy. In the present study, the porcine parthenotes originated from electric stimulation implanted and developed normally during the first month, in a manner similar to artificially inseminated embryos and fetuses. There were no statistical differences in the formation of the major organs such as the brain, liver, kidney, or heart in both groups. However, the implanted parthenotes radically ceased their development and degenerated after one month. It can be postulated that the parthenotes are one of the reasons for the gap between early pregnancy and delivery rate in assisted reproduction techniques. Abstract The difference between early pregnancy and delivery rate is quite large in assisted reproduction techniques (ARTs), including animal cloning. However, it is not clear why the implanted fetuses aborted after the early pregnancy stage. In the present study, we tried to evaluate the developmental and morphological characteristics of porcine parthenogenetically activated (PA) embryos or fetuses by electric stimulation during the early pregnancy period. The implanted PA and artificially inseminated (AI) embryos and fetuses were collected at day 26 and 35 after embryo transfer, respectively. The developmental and morphological parameters in the PA embryos at day 26 were similar to the AI embryos. The size, weight, formation of major organs, and apoptotic cells were not statistically different in both embryos at day 26. However, the PA fetuses at day 35 showed ceased fetal development and degenerated with abnormal morphologies in their organs. The day 35 PA fetuses showed significantly higher apoptotic cells and lower methylation status in three differentially methylated regions of the H19 gene compared to their comparators. Therefore, the normal development of PA embryos and fetuses during early gestation could lead to these pregnancies being misinterpreted as normal and become one of the main reasons for the gap between early pregnancy and delivery rate.

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B&ndash, Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

We developed a fully closed nano-focus X-ray source using carbon nanotube (CNT) field emitters. The nano-focus X-ray source includes a fully vacuum-sealed CNT Electron-gun (E-gun) tube and E-beam-focusing modules consisting of electrostatic and magnetic lenses, facilitating very compact and high-resolution X-ray imaging. The paste-printed CNT emitters were prepared in a small pattern as an electron source, and the entire components, including the CNT emitter module, Cu tubes, collimators, and transmissive anode target, were specially brazed at an elevated temperature, resulting in a very compact E-gun tube. The CNT emitter showed a stable and reliable emission current with its density of over 300 mA/cm2 even in the vacuum-sealed E-gun tube. With the anode grounded and the CNT emitter module biased to negative high voltages, we achieved a considerably magnified x-ray image of a several hundred nanometer resolution. The fully vacuum-sealed CNT E-gun tube along with the magnetic lens module can offer much more compact, easily maintainable, high-resolution X-ray imaging compared with conventional open-type thermionic X-ray sources.

Alternative splicing and alternative promoter usage have been shown to have an integral role in creating flexibility in the regulation of gene expression. Previous studies collectively showed that expression of the retinol binding protein 7 (Rbp7) gene was adipose tissue-specific across species. Nevertheless, alternative splicing and alternative promoter usage of the Rbp7 gene in adipose tissue and other tissues have not been investigated. The objectives of this study were to investigate protein isoforms of RBP7 produced from alternative splicing, alternative promoter usage and pre-mRNA trans-splicing, and to examine expression patterns of RBP7 isoforms during adipogenesis, cold exposure, and retinol or retinoic acid treatment. Our RT-PCR analysis revealed that mouse Rbp7 isoforms were present, but only one protein isoform was detected which was specific to adipose tissue. In addition, a fusion transcript of the Nmnat1 gene and the Rbp7 gene was produced by pre-mRNA trans-splicing in several tissues; however, its protein expression was not detectable. During adipogenesis, RBP7 expression was prominent in both neonatal and after-weaning stages and its expression was significantly higher in fat cells than in preadipocytes. Exposure to cold led to an increased expression of RBP7 in brown adipose tissue (BAT). Furthermore, Rbp7 mRNA expression in 3T3-L1 cells was significantly up- and down-regulated by retinol and retinoic acid, respectively. Our data showed that the mouse Rbp7 gene produces a predominant isoform in adipose tissue during adipocyte development, cold exposure, and nutritional treatments, which can be a potential target for future investigation on reduced adiposity.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.

Although a focused electron beam is required in field emission-based various applications, the electron beam is generally diverged in a conventional triode because the gate has a circular aperture through which the electrons are accelerated to reach the anode. To prevent the beam divergence, a suspended multi-layer graphene (MLG) was fabricated on a very large gate aperture to be used as an electron-transparent gate in a field emission triode. The suspended MLG was observed to have several micro-openings with an average diameter of ∼5 μm formed during a transfer. It was observed that the MLG with micro-openings was likely to act as an efficient transparent gate for electrons even with large number of graphene layers, exhibiting a high transmittance of ∼90%. The solid angles of the beams obtained using conventional gate aperture and suspended MLG gate were 0.225 and 0.074 sr, respectively, indicating that the suspended MLG resulted in a focused electron beam. Simulations confirm that the suspended MLG with micro-openings suppresses the beam divergence in a given triode configuration because the MLG results in the flattened electric field profiles between gate and cathode whereas a distorted potential distribution causing beam divergence occurs around the aperture in a conventional triode.

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B–Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

Ice-binding proteins (IBPs) facilitate organism survival under extreme conditions by inhibiting thermal hysteresis and ice recrystallization. IBPs have been widely used as cryoprotectants to cryopreserve mammalian gametes and embryos. In the present study, we evaluated the protective effects of an Arctic yeast, Leucosporidium sp. AY30 derived ice-binding protein (LeIBP), on the vitrification of bovine metaphase II (MII) oocytes and embryos. When oocytes and embryos were frozen using the two-step vitrification method, the survival rate was significantly increased in the presence of LeIBP. The LeIBP supplementation decreased the levels of intracellular reactive oxygen species (ROS) and enhanced mitochondrial functions in the vitrified-warmed oocytes. Furthermore, LeIBP improved the developmental potential and suppressed apoptosis of the embryos derived from vitrified-warmed oocytes. Collectively, these data indicate that LeIBP can be used as a promising cryoprotectant to prevent cryoinjury during vitrification in bovine oocytes.

The glycosylphosphatidylinositol-anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either

Pluripotent stem cells can be established from parthenogenetic embryos, which only possess maternal alleles with maternal-specific imprinting patterns. Previously, we and others showed that parthenogenetic embryonic stem cells (pESCs) and parthenogenetic induced pluripotent stem cells (piPSCs) progressively lose the bimaternal imprinting patterns. As ESCs and iPSCs are na&iuml, ve pluripotent stem cells, parthenogenetic primed pluripotent stem cells have not yet been established, and thus, their imprinting patterns have not been studied. Here, we first established parthenogenetic epiblast stem cells (pEpiSCs) from 7.5 dpc parthenogenetic implantation embryos and compared the expression patterns and DNA methylation status of the representative imprinted genes with biparental EpiSCs. We found that there were no striking differences between pEpiSCs and biparental EpiSCs with respect to morphology, pluripotency gene expression, and differentiation potential, but there were differences in the expression and DNA methylation status of imprinted genes (H19, Igf2, Peg1, and Peg3). Moreover, pEpiSCs displayed a different DNA methylation pattern compared with that of parthenogenetic neural stem cells (pNSCs), which showed a typical bimaternal imprinting pattern. These results suggest that both na&iuml, ve pluripotent stem cells and primed pluripotent stem cells have an unstable imprinting status.

Spermatogenesis is a process by which haploid cells differentiate from germ cells in the seminiferous tubules of the testes. TLE3, a transcriptional co-regulator that interacts with DNA-binding factors, plays a role in the development of somatic cells. However, no studies have shown its role during germ cell development in the testes. Here, we examined TLE3 expression in the testes during spermatogenesis. TLE3 was highly expressed in mouse testes and was dynamically regulated in different cell types of the seminiferous tubules, spermatogonia, spermatids, and Sertoli cells, but not in the spermatocytes. Interestingly, TLE3 was not detected in Sertoli cells on postnatal day 7 (P7) but was expressed from P10 onward. The microarray analysis showed that the expression of numerous genes changed upon TLE3 knockdown in a Sertoli cell line TM4. These include 1597 up-regulated genes and 1452 down-regulated genes in TLE3-knockdown TM4 cells. Ingenuity Pathway Analysis (IPA) showed that three factors were up-regulated and two genes were down-regulated upon TLE3 knockdown in TM4 cells. The abnormal expression of the three factors is associated with cellular malfunctions such as abnormal differentiation and Sertoli cell formation. Thus, TLE3 is differentially expressed in Sertoli cells and plays a crucial role in regulating cell-specific genes involved in the differentiation and formation of Sertoli cells during testicular development.

During embryonic development, cells undergo changes in gene expression, signaling pathway activation/inactivation, metabolism, and intracellular organelle structures, which are mediated by mitochondria. Mitochondria continuously switch their morphology between elongated tubular and fragmented globular via mitochondrial fusion and fission. Mitochondrial fusion is mediated by proteins encoded byMfn1,Mfn2, andOpa1, whereas mitochondrial fission is mediated by proteins encoded byFis1andDnm1L. Here, we investigated the expression patterns of mitochondria-related genes during the differentiation of mouse embryonic stem cells (ESCs). Pluripotent ESCs maintain stemness in the presence of leukemia inhibitory factor (LIF) via the JAK-STAT3 pathway but lose pluripotency and differentiate in response to the withdrawal of LIF. We analyzed the expression levels of mitochondrial fusion- and fission-related genes during the differentiation of ESCs. We hypothesized that mitochondrial fusion genes would be overexpressed while the fission genes would be downregulated during the differentiation of ESCs. Though the mitochondria exhibited an elongated morphology in ESCs differentiating in response to LIF withdrawal, only the expression ofMfn2was increased and that ofDnm1Lwas decreased as expected, the other exceptions beingMfn1,Opa1, andFis1. Next, by comparing gene expression and mitochondrial morphology, we proposed an index that could precisely represent mitochondrial changes during the differentiation of pluripotent stem cells by analyzing the expression ratios of three fusion- and two fission-related genes. Surprisingly, increasedMfn2/Dnm1Lratio was correlated with elongation of mitochondria during the differentiation of ESCs. Moreover, application of this index to other specialized cell types revealed that neural stems cells (NSCs) and mouse embryonic fibroblasts (MEFs) showed increasedMfn2/Dnm1Lratio compared to ESCs. Thus, we suggest that theMfn2/Dnm1Lratio could reflect changes in mitochondrial morphology according to the extent of differentiation.

Data Availability Statement: Full-length MYH3 CDS sequence from KNP, KX538787; full-length MYH3 CDS sequence from Landrace, KX538788. Two-kb of 5’-UTR MYH3 genomic DNA sequence from KNP, KX549312; 0.5-kb of 3’-UTR MYH3 genomic DNA sequence from KNP, KX549313. Two-kb of 5’-UTR MYH3 genomic DNA sequence from Landrace, KX549311; 0.5-kb of 3’-UTR MYH3 genomic DNA sequence from Landrace, KX549314. Resequencing data access information is provided in S10 Table. All the raw data to produce figures and tables are available at https://datadryad.org/review?doi=doi:10.5061/dryad.dr32n87, Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5'-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs., This work was carried out with the support of >Cooperative Research Program for Agriculture Science & Technology Development (Project title: A research on performance improvement of ‘Nanchukmacdon’, a high quality pig breed developed using Korean native black pigs (KNP) in Jeju Island, Project No. PJ01012301 and PJ01262701)> Rural Development Administration (http://www.rda.go.kr), Republic of Korea. This work was also supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program, Republic of Korea (www.kribb.re.kr). M.P.E work was funded by AGL2016-78709-R (Ministerio de Economía y Competitividad, Spain; http://www.mineco.gob.es/portal/site/mineco/). T

Adhesion of carbon nanotube (CNT) onto a cathode substrate is very crucial for field electron emitters that are operating under high electric fields. As a supporting precursor of CNT field emitters, we adopted silicon carbide (SiC) nano-particle fillers with Ni particles and then enhanced interfacial reactions onto Kovar-alloy substrates through the optimized wet pulverization process of SiC aggregates for reliable field electron emitters. As-purchased SiC aggregates were efficiently pulverized from 20 to less than 1 micro-meter in a median value (D50). CNT pastes for field emitters were distinctively formulated by a mixing process of the pulverized SiC aggregates and pre-dispersed CNTs. X-ray photoelectron spectroscopy studies showed that the optimally pulverized SiC-CNT paste-emitter had a stronger Si 2p3/2 signal in the Ni2Si phase than the as-purchased one. The Si 2p3/2 signal would represent interfacial reaction of the SiC nano-particle onto Ni from the CNT paste and the Kovar substrate, forming the supporting layer for CNT emitters. The optimal paste-emitter even in a vacuum-sealed tube exhibited a highly reliable field emission current with a high current density of 100 mA cm−2 for over 50 h along with good reproducibility. The enhanced interfacial reaction of SiC filler onto the metal substrates could lead to highly reliable field electron emitters for vacuum electronic devices.

Summary Naive and primed pluripotent stem cells (PSCs) and germ cells express the Oct4 gene. The Oct4 gene contains two cis-regulatory elements, the distal enhancer (DE) and proximal enhancer (PE), which differentially control Oct4 expression in a cell-type-specific and stage-specific manner. Here, we generated double transgenic mice carrying both Oct4-ΔPE-GFP and Oct4-ΔDE-tdTomato (RFP), enabling us to simultaneously monitor the activity of DE and PE. Oct4 expression is stage-specifically regulated by DE and PE during embryonic and germ cell development. Using this dual reporter system, we successfully cultured pure populations of naive (GFP+RFP−) and primed (GFP−RFP+) PSCs. We found that GFP+RFP− cells were metastable (not naive) in serum-containing medium; stable naive pluripotent cells were observed in medium containing two inhibitors (Meki and GSKi) but lacked serum. Finally, we suggest that the activity of Oct4 DE and PE is regulated by the repressive histone marks and DNA methylation in a cell-type-specific manner., Graphical Abstract, Highlights • A defined model for Oct4 enhancer activity in the totipotent cycle • Culturing pure populations of naive and primed PSCs by a double reporter system • Altering Oct4 enhancer activity in PSCs by changing culture conditions • Histone modification and DNA methylation regulate Oct4 enhancer activity, Do and colleagues show that Oct4 expression is stage-specifically regulated by enhancer activity during embryonic and germ cell development by using a dual reporter system. Enhancer activity is altered in pluripotent stem cells by culture environment, which is regulated by histone modification and DNA methylation.

Pigs have been used as a good research model for xenotransplantation. Several groups have generated porcine-induced pluripotent stem cells (piPSCs) from differentiated somatic cells. Transgenic pigs with the alpha1,3-galactosyltransferase gene-knockout (GalT-KO) could successfully govern hyper acute rejection of organ transplants into primates. Thus, GalT-KO piPSCs could be a powerful cell resource for agricultural and biomedical applications. This study was performed to generate iPSCs from GalT-KO pigs and characterize their properties. We successfully generated a GalT-KO iPSC from a genetically modified pig using double alpha1,3-galactosyltransferase knockout alterations. Similar to mouse embryonic stem cells, the GalT-KO piPSCs were positive for classical pluripotency markers: POU5F1, NANOG, SOX2 and SSEA1, and were negative for: SSEA3, TRA-1-60 and TRA-1-81. Furthermore, these cells could form an embryoid body that differentiated into three germ layers in vitro, and were highly proliferative u...

Premature ovarian failure (POF) is a major side effect of chemotherapy in young cancer patients. To develop pharmaceutical agents for preserving fertility, it is necessary to understand the mechanisms responsible for chemotherapy-induced follicle loss. Here, we show that treatment with cisplatin, a widely used anticancer drug, depleted the dormant follicle pool in mouse ovaries by excessive activation of the primordial follicles, without inducing follicular apoptosis. Moreover, we show that co-treatment with the antioxidant melatonin prevented cisplatin-induced disruption of the follicle reserve. We quantified the various stages of growing follicles, including primordial, primary, secondary, and antral, to demonstrate that cisplatin treatment alone significantly decreased, whereas melatonin co-treatment preserved, the number of primordial follicles in the ovary. Importantly, analysis of the PTEN/AKT/FOXO3a pathway demonstrated that melatonin significantly decreased the cisplatin-mediated inhibitory phosphorylation of PTEN, a key negative regulator of dormant follicle activation. Moreover, melatonin prevented the cisplatin-induced activating phosphorylation of AKT, GSK3β, and FOXO3a, all of which trigger follicle activation. Additionally, we show that melatonin inhibited the cisplatin-induced inhibitory phosphorylation and nuclear export of FOXO3a, which is required in the nucleus to maintain dormancy of the primordial follicles. These findings demonstrate that melatonin attenuates cisplatin-induced follicle loss by preventing the phosphorylation of PTEN/AKT/FOXO3a pathway members; thus, melatonin is a potential therapeutic agent for ovarian protection and fertility preservation during chemotherapy in female cancer patients.

Vitamin A, referred to as retinol, is an essential nutrient that affects the cell growth and differentiation including adipogenesis. Although previous studies using supraphysiological doses (over 1 μM) of all-trans-retinoic acid (atRA) demonstrated antiadipogenic activity, effects of atRA at various levels on differentiation of 3T3-L1 preadipocytes have not been extensively investigated. Our study showed that the amount of cellular triacylglycerol (TAG) and intensities of Oil-Red-O staining were decreased by supplementing atRA (1 and 10 μM) but increased by low concentrations of atRA (0.01 to 100 nM) compared with the control. Also PPARγ and FABP4 were gradually overexpressed by atRA up to 1 nM but decreased at over 1 nM concentrations. Moreover, mitotic clonal expansion (MCE) and consequential growth-arrest were analyzed as important steps in adipogenesis of 3T3-L1 cells. The 1 nM group showed more cell proliferation and thereafter a higher ratio of the G0/G1 phase on Day 2. Protein levels of S/G2-phase factors were dose dependently increased by atRA up to 1 nM on Day 1, but the factors were highly expressed in higher doses on Day 2. G0/G1 markers were higher at the higher doses of atRA on Day 1; whereas, they were highly expressed in mild or medium doses on Day 2. These data indicate that atRA controls adipogenesis with accompanied changes in cell proliferation and follow-up growth-arrest. These results indicate that atRA can function both as a negative and positive regulator of adipogenesis depending on dosages, providing a strategy for achieving proper nutritional balance for treatment of obesity.

We improved carbon nanotube (CNT) paste field emitter by optimizing the size distribution of SiC aggregates as inorganic fillers using a novel milling process. The average size of SiC aggregates was reduced to sub-micrometer with a relatively narrow distribution. The CNT paste emitter with the optimized small SiC fillers of 1 m in D50, by the milling process, exhibited better adhesive property and more uniform surface morphology than that with large fillers from the as-purchased SiC. Furthermore, the optimized small fillers shows superior field emission performance even under a high current density, making the CNT paste technology very useful for a variety of field emission devices.

The printed carbon nanotube (CNT) paste emitters were applied to two types of vacuum-sealed x-ray tubes for several hundred-nanometer-resolution non-destructive testing (NDT) and cone-beam breast computed tomography (CB-BCT) applications. A single dot CNT emitter of 200 micrometer in diameter was made for a small focal spot size of NDT imaging meanwhile a 983-dots array was for a high current of CB-BCT imaging. The electron beam emitted from the single dot emitter in the nano-focus x-ray tube is strongly focused to a small focal spot size of 1 micrometer or less in diameter by help of electrostatic and magnetic focusing lenses, and the 983 emitters produce a large field emission current over 200 mA with a moderate focal spot, tentatively applicable to CB-BCT imaging. The printed CNT emitters can be used for a variety of electron sources from several hundred-nanometer focusing to high-flux xray tubes.

Multilayer graphene-gated carbon nanotube (CNT) emitters are introduced to novel vacuum-sealed x-ray tubes. We characterized field emission performances and electron-beam (Ebeam) converging of the graphene-gated emitter in a vacuum chamber. Thermal durability of the graphene-gate was well achieved to apply it to x-ray tubes processed with brazing at a high temperature. A transmissive anode target with a Be window, ceramic insulators, and the graphene-gated CNT emitters were successfully brazed to a fully vacuum-sealed x-ray tube, maintaining an unique graphene-gated field emission performance.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aarssti mutant mice resulted in an increased production of serine-mischarged tRNAAla and degeneration of cerebellar Purkinje cells. By positional cloning, we identified Ankrd16, which acts epistatically with the Aarssti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific, ankyrin repeat-containing protein, binds directly to the catalytic domain of AlaRS. Serine misactivated by AlaRS is captured by lysine side chains of ANKRD16, preventing the charging of serine adenylates to tRNAAla and precluding serine misincorporation in nascent peptides. Deletion of Ankrd16 in the Aarssti/sti brain causes widespread protein aggregation and neuron loss. These results identify a novel amino acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery essential for preventing severe pathologies that arise from defects in editing.

Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) in H19 was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such as GAPDH and β-actin were unchanged in the serially cloned pigs. In addition, expression levels of H19 and Igf2 were variable for each generation of serial SCNT piglets, but there was no clear relationship between the meth...

We propose an advanced active current-control (ACC) unit composed of two cascade transistors for extremely stabilizing field emission currents. The upper transistor connected to the cathode modulates high voltages for field emission while the other one regulates emission current stably. Using the ACC unit developed, very stable emission currents were achieved from the fully vacuum-sealed x-ray tube with carbon nanotube field emitters for long pulse sequence. The advanced ACC unit could provide commercial stability and reliability of field emission x-ray sources even in high voltage and/or high duty environments.

We tried to improve field emission properties of carbon nanotube (CNT) paste emitters by controlling the size distribution of aggregated SiC fillers. The SiC fillers used as an adhesion promoter strongly affects the dispersion of CNT paste, determining uniformity and reproducibility of the field emitter. We found, to date, that the optimum distribution is about 1 μm of D50, the value of the particle diameter at 50% in cumulative distribution. The CNT emitters with the optimized paste showed uniform and reproducible emission performances along with high adhesion to the cathode. The improved paste formulation and emitter fabrication could lead to successful field emission devices and applications.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars

The Kit gene is proto-oncogene, encoded stem cell factor and involved in melanocyte development. Also, Kit is expressed in epidermis and proliferation of melanoblasts which occur to pigmentation in skin, eyes and other tissues. Here we established porcine Kit genes overexpressed transgenic mice. The role of Kit gene was regulation in melanogensis remains unclear in porcine. Therefore, present study aims to ectopic expression porcine Kit gene in pigmentation of skin tissues. Our results showed that no effect on melanogenesis in ectopic Kit gene overexpressed transgenic mice. Further studies on Kit gene will provide insight into other specific roles in porcine and mice species.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p